Biological Activity

Bioactivity / Mechanism

Flumazenil (Ro 15-1788) shows condition-dependent benzodiazepine-site pharmacology. It antagonized midazolam’s discriminative stimulus in rhesus monkeys, while recombinant-receptor recordings detected weak potentiation of GABA currents in several receptor subtypes. Its activity should therefore be interpreted in the specific receptor and drug context. [1,2]

Literature-reported observations and conditions; not Adooq-tested protocols or general treatment recommendations.

Target & Activity

Target	Activity	Value
GABAA receptor

Cellular Effect

Cell Line	Activity	Value	Description	Reference
Oocyte	EC50	30 nM	Activity at rat wild type GABAA alpha-1H101Cbeta-2-gamma-2 mutant receptor expressed in xenopus oocytes assessed as GABA-elicited response by two electrode voltage clamp method relative to control	[PMID: 17626010]

In Vitro Studies

In cultured rat hippocampal neurons exposed to flurazepam, acute Ro 15-1788 restored surface receptor cluster area/fluorescence and mIPSC amplitude toward control levels. Cluster density did not increase significantly after rescue, and mIPSC frequency was reported unaffected across conditions. [3]

Subtype-dependent modulation of recombinant GABAA receptors

Cell-based study · Xenopus oocytes expressing rat α1–6β3γ2 GABAA receptors [1]

Flumazenil weakly potentiated GABA currents in α2-, α3-, α4- and α6-containing receptors; no significant modulation was detected in α1β3γ2 or α5β3γ2.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Potentiation of GABA EC3 current in α2β3γ2 and α3β3γ2 receptors	EC50 α2β3γ2: 5 ± 1 nM; α3β3γ2: 11 ± 5 nM	[1]
Current at 10 μM Ro15-1788, normalized to GABA EC3 alone (100%)	% control current α2β3γ2: 133 ± 7%; α3β3γ2: 157 ± 8%; α4β3γ2: 181 ± 10%; α6β3γ2: 179 ± 6%	[1]
Low-potency subtypes and absence of significant intrinsic modulation	EC50 / significance α4β3γ2: >232 nM; α6β3γ2: >204 nM; α1β3γ2 and α5β3γ2: ns at listed 10 nM, 100 nM, 1 μM and 10 μM	[1]

Literature-Reported Treatment Conditions

Treatment concentration: 1 nM–10 μM concentration–effect curves
Exposure duration: 30 s drug preapplication, then coapplication with GABA until peak response
Method: Two-electrode voltage clamp; GABA EC3, −60 mV, room temperature
Vehicle: 0.1% DMSO in XR solution
Co-treatment: GABA titrated to EC3 in each oocyte
Reported limits: No Adooq material or lot equivalence established.

Methods and source locations

Compound reported in the paper: Flumazenil; Compound label in the paper: Ro15-1788

Findings apply to the reported material and model; they do not validate an Adooq lot.

Section 3.1, Ro15-1788 paragraph; Table 1, Ro15-1788 rows; Figure 4A and caption; Sections 2.1–2.2

Potentiation of GABA EC3 current in α2β3γ2 and α3β3γ2 receptors: Functional concentration–effect fits; mean ± SEM; Figure 4 data from at least 3 oocytes derived from ≥2 batches. — Section 3.1, Ro15-1788 paragraph; Table 1, Ro15-1788 rows; Figure 4A and caption; Sections 2.1–2.2

Current at 10 μM Ro15-1788, normalized to GABA EC3 alone (100%): Table 1; mean ± SEM; significant values differ from GABA EC3, Student t-test, at least P < 0.05. — Section 3.1, Ro15-1788 paragraph; Table 1, Ro15-1788 rows; Figure 4A and caption; Sections 2.1–2.2

Low-potency subtypes and absence of significant intrinsic modulation: Original table operators retained; ns means not significant, not zero effect or an antagonism assay. — Section 3.1, Ro15-1788 paragraph; Table 1, Ro15-1788 rows; Figure 4A and caption; Sections 2.1–2.2

Rescue of surface receptor clusters after flurazepam exposure

Cell-based study · Embryonic-day-18 rat hippocampal neurons expressing α2pHGFP; imaging at 14–17 DIV [3]

After flurazepam exposure, acute Ro 15-1788 restored surface cluster area and fluorescence toward control levels. Cluster density was not significantly increased.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Mean surface α2pHGFP cluster area	μm² Control: 0.232 ± 0.008; Flz: 0.184 ± 0.006; Flz + Ro: 0.216 ± 0.010; control + Ro: 0.242 ± 0.006	[3]
Total surface cluster fluorescence	% control Control: 101.4 ± 5.4; Flz: 73.4 ± 3.6; Flz + Ro: 96.5 ± 5.3; control + Ro: 104.9 ± 5.4	[3]
Surface cluster density after rescue treatment	clusters per 20 μm Control: 6.8 ± 2.2; Flz: 5.4 ± 1.2; control + Ro: 6.9 ± 1.8; Flz + Ro: 5.4 ± 1.6	[3]

Literature-Reported Treatment Conditions

Treatment concentration: 5 μM Ro 15-1788 after 250 nM flurazepam
Exposure duration: 24 h flurazepam or control, followed by 1 h Ro 15-1788 immediately before imaging
Method: Live-cell confocal imaging at 37 °C in HBS
Co-treatment: Flurazepam (Flz) pretreatment; control ± Ro 15-1788 and Flz ± Ro 15-1788 comparisons
Reported limits: Treatment stock vehicle not identified in the read main text; SI was not retrieved.

Methods and source locations

Compound reported in the paper: Flumazenil; Compound label in the paper: Ro 15-1788

Findings apply to the reported material and model; they do not validate an Adooq lot.

Results, rescue of surface GABAA receptor levels; Figure 2A–B and caption; Materials and Methods, cell culture/chemicals/live imaging

Mean surface α2pHGFP cluster area: Mean ± SEM; 22–30 neurons, n = 3 cultures; Figure 2B uses one-way ANOVA and Bonferroni multiple comparisons, *P < 0.05. — Results, rescue of surface GABAA receptor levels; Figure 2A–B and caption; Materials and Methods, cell culture/chemicals/live imaging

Total surface cluster fluorescence: Mean ± SEM; 22–30 neurons, n = 3 cultures. Exact values from Results, not estimated from bars. — Results, rescue of surface GABAA receptor levels; Figure 2A–B and caption; Materials and Methods, cell culture/chemicals/live imaging

Surface cluster density after rescue treatment: Results state Ro did not significantly increase cluster density in Flz-treated neurons. Control + Ro showed no significant change in measured parameters; uncertainty follows the figure mean ± SEM convention. — Results, rescue of surface GABAA receptor levels; Figure 2A–B and caption; Materials and Methods, cell culture/chemicals/live imaging

Recovery of inhibitory current amplitude after flurazepam exposure

Cell-based study · Rat hippocampal neurons cultured 2–3 weeks; whole-cell miniature inhibitory postsynaptic currents (mIPSCs) [3]

Ro 15-1788 returned flurazepam-reduced mIPSC amplitude to control levels. Frequency, rise time and decay time were not significantly altered under the reported conditions.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Mean mIPSC amplitude	pA Control: 52.5 ± 2.7; Flz: 42.2 ± 2.4; Flz + Ro: 50.2 ± 5.7	[3]
mIPSC frequency	Hz Control: 1.08 ± 0.34; Flz: 0.69 ± 0.12; Flz + Ro: 0.68 ± 0.10	[3]

Literature-Reported Treatment Conditions

Treatment concentration: 5 μM Ro 15-1788 after 250 nM flurazepam
Exposure duration: 24 h flurazepam; 1 h Ro 15-1788 before recording
Method: Whole-cell mIPSC recording
Co-treatment: Untreated control, Flz, and Flz + Ro comparisons
Reported limits: Stock vehicle and additional SI recording details were not retrieved. No separate Ro-only electrophysiology arm is shown in Figure 2C–D.

Methods and source locations

Compound reported in the paper: Flumazenil; Compound label in the paper: Ro 15-1788

Findings apply to the reported material and model; they do not validate an Adooq lot.

Results, Prolonged Exposure of Neurons to BZ Reduces mIPSCs; Figure 2C–D and caption; Materials and Methods, chemicals/electrophysiology

Mean mIPSC amplitude: Mean ± SEM; n = 7–10 cells per condition; Figure 2D: *P < 0.05, Kolmogorov–Smirnov test. Results describe rescue to control levels. — Results, Prolonged Exposure of Neurons to BZ Reduces mIPSCs; Figure 2C–D and caption; Materials and Methods, chemicals/electrophysiology

mIPSC frequency: Mean ± SEM; n = 7–10 cells per condition; reported unaffected. Rise and decay times also reported unaffected; no exact values inferred from traces. — Results, Prolonged Exposure of Neurons to BZ Reduces mIPSCs; Figure 2C–D and caption; Materials and Methods, chemicals/electrophysiology

In Vivo Studies

In a four-monkey discrimination cohort, 0.1 mg kg−1 subcutaneous flumazenil shifted the midazolam ED50 9.8-fold higher but the alfaxolone ED50 3.0-fold lower. The opposite interactions show why benzodiazepine antagonism should not be generalized to other GABAA receptor modulators. [2]

Contrasting interactions with midazolam and alfaxolone in rhesus monkeys

Animal study · Adult rhesus monkeys trained to discriminate subcutaneous midazolam from saline; Figure 3 cohort n = 4 of 5 trained monkeys [2]

Flumazenil attenuated the midazolam discriminative stimulus but enhanced the midazolam-like effect of alfaxolone in the same four monkeys. These are drug-interaction effects, not flumazenil monotherapy efficacy.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Midazolam dose producing 50% drug-lever responding	ED50 dose ratio 9.8-fold increase with 0.1 mg kg−1 flumazenil	[2]
Alfaxolone dose producing 50% midazolam-lever responding	ED50 dose ratio 3.0-fold decrease with 0.1 mg kg−1 flumazenil	[2]
Operant response-rate control	qualitative 0.1 mg kg−1 flumazenil alone did not decrease response rate; rate decreased in 1 of 4 monkeys when combined with the largest tested alfaxolone dose (3.2 mg kg−1).	[2]

Conditions shared by the study groups

Route: s.c.
Frequency: One pretreatment per cumulative-dose test session
Vehicle: 40% propylene glycol, 50% saline, 10% ethanol

Study groups and reported outcomes
Group / dose	Comparator	Observed result	Reference
Flumazenil plus midazolam Flumazenil 0.1 mg kg−1	midazolam dose–response without flumazenil; vehicle/control response rates	Midazolam ED50 increased 9.8-fold.	[2]
Flumazenil plus alfaxolone Flumazenil 0.1 mg kg−1	alfaxolone dose–response without flumazenil; vehicle/control response rates	Alfaxolone ED50 decreased 3.0-fold; response-rate suppression occurred in 1 of 4 at the largest tested alfaxolone dose.	[2]

Flumazenil plus midazolam: Model: Adult rhesus monkeys; Figure 3, n = 4; Treatment course: 15 min before first midazolam dose; cumulative test doses thereafter

Flumazenil plus alfaxolone: Model: Adult rhesus monkeys; Figure 3, n = 4; Treatment course: 15 min before first alfaxolone dose; cumulative test doses thereafter

Methods and source locations

Compound reported in the paper: Flumazenil; Compound label in the paper: flumazenil

Findings apply to the reported material and model; they do not validate an Adooq lot.

Methods, subjects/midazolam discrimination/drugs/data analyses; Results, Figure 3 and caption, with Figure 1 axis conventions; fifth-monkey paragraph

Midazolam dose producing 50% drug-lever responding: Results exact dose ratio; n = 4, Figure 3. Graphical means ± SEM; no numerical uncertainty reported for this ratio. — Methods, subjects/midazolam discrimination/drugs/data analyses; Results, Figure 3 and caption, with Figure 1 axis conventions; fifth-monkey paragraph

Alfaxolone dose producing 50% midazolam-lever responding: Results exact dose ratio; same n = 4, Figure 3. This enhancement is an important limit to a universal antagonist description. — Methods, subjects/midazolam discrimination/drugs/data analyses; Results, Figure 3 and caption, with Figure 1 axis conventions; fifth-monkey paragraph

Operant response-rate control: Results accompanying Figure 3, lower panels. Discrimination data excluded when rate <20% of individual control; rate data retained. — Methods, subjects/midazolam discrimination/drugs/data analyses; Results, Figure 3 and caption, with Figure 1 axis conventions; fifth-monkey paragraph

Flumazenil plus midazolam · Methods, subjects/midazolam discrimination/drugs/data analyses; Results, Figure 3 and caption, with Figure 1 axis conventions; fifth-monkey paragraph

Flumazenil plus alfaxolone · Methods, subjects/midazolam discrimination/drugs/data analyses; Results, Figure 3 and caption, with Figure 1 axis conventions; fifth-monkey paragraph

Specifications

Manufacturer	Adooq Bioscience
Actions	Antagonist
CAS Number	78755-81-4
Applications	Benzodiazepine antagonist
Formula	C₁₅H₁₄FN₃O₃
Molecular Weight	303.3
Synonyms	Ro 15-1788
SMILES	CCOC(=O)c1ncn2c1CN(C)C(=O)c1c2ccc(c1)F

Storage & Solubility

In Vitro Solubility

Reported Solubility

Solvent	Reported Solubility
DMSO	4 mg/mL (13.18 mM)
Water	Insoluble
Ethanol	Insoluble

Preparing Stock Solutions

Stock preparation is unavailable for this product molecular weight.

Calculated stock concentrations do not establish solvent solubility. Refer to the reported solubility data for experimental guidance.

Storage

Store powder at -20°C, keep desiccated.
In powder form, the chemical is stable for 36 months.
In solution, store at -20°C and use within 1 month to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles.

Research References

[1] Ramerstorfer J, Furtmüller R, Vogel E, et al. The point mutation gamma 2F77I changes the potency and efficacy of benzodiazepine site ligands in different GABAA receptor subtypes. European journal of pharmacology (2010). PMID: 20303942 DOI: 10.1016/j.ejphar.2010.03.015 Source material
[2] McMahon LR, France CP. Differential behavioral effects of low efficacy positive GABAA modulators in combination with benzodiazepines and a neuroactive steroid in rhesus monkeys. British journal of pharmacology (2006). PMID: 16331290 DOI: 10.1038/sj.bjp.0706550 Source material
[3] Jacob TC, Michels G, Silayeva L, et al. Benzodiazepine treatment induces subtype-specific changes in GABA(A) receptor trafficking and decreases synaptic inhibition. Proceedings of the National Academy of Sciences of the United States of America (2012). PMID: 23091016 DOI: 10.1073/pnas.1204994109 Source material

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Flumazenil

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Flumazenil 5 mg

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Biological Activity

Bioactivity / Mechanism

Target & Activity

Cellular Effect

In Vitro Studies

Subtype-dependent modulation of recombinant GABAA receptors

Literature-Reported Treatment Conditions

Rescue of surface receptor clusters after flurazepam exposure

Literature-Reported Treatment Conditions

Recovery of inhibitory current amplitude after flurazepam exposure

Literature-Reported Treatment Conditions

In Vivo Studies

Contrasting interactions with midazolam and alfaxolone in rhesus monkeys

Conditions shared by the study groups

Specifications

Storage & Solubility

In Vitro Solubility

Reported Solubility

Preparing Stock Solutions

Storage

Research References

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